A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover.

Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance.

AMP-Activated Protein Kinase α2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1α and a Network of Proteins Affecting Metabolism and Apoptosis.

Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.

Objective: To determine the role of the AMPKα2 subunit in vascular repair.

Methods And Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2 versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2).

Advanced light microscopy core facilities: Balancing service, science and career.

Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models.

Cytotoxicity and infiltration of human NK cells in in vivo-like tumor spheroids.

Background: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.

Methods: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening.

Live spheroid formation recorded with light sheet-based fluorescence microscopy.

We provide a detailed protocol for a three-dimensional long-term live imaging of cellular spheroids with light sheet-based fluorescence microscopy. The protocol allows the recording of all phases of spheroid formation in three dimensions, including cell proliferation, aggregation, and compaction. We employ the human hepatic cell line HepaRG transfected with the fusion protein H2B-GFP, i.

Quantifying the autophagy-triggering effects of drugs in cell spheroids with live fluorescence microscopy.

We present a 3D assay for the quantification of the autophagic flux in live cell spheroids by using the fluorescent reporter mRFP-GFP-LC3. The protocol describes the formation of the spheroids from the astrocytoma cell line U343, live long-term 3D fluorescence imaging of drug-treated spheroids, and the image processing workflow required to extract quantitative data on the autophagic flux.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs.

Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells.

Cadherins, Ca(2+)-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca(2+)-dependent interactions.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways.

High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy.

Conventional two-dimensional cell monolayers do not provide the geometrical, biochemical and mechanical cues found in real tissues. Cells in real tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Such a highly interconnected communication network extends along all three dimensions.

Quantitative 3D cell-based assay performed with cellular spheroids and fluorescence microscopy.

Cell-based assays are essential in both basic research and drug discovery. Three-dimensional cellular spheroids are more realistic models of tumors and healthy tissues compared to standard two-dimensional cultures. Employing spheroids improves the reliability and the physiological significance of cell-based assays.

Impact of melatonin and molecular clockwork components on the expression of thyrotropin beta-chain (Tshb) and the Tsh receptor in the mouse pars tuberalis.

Photoperiodic regulation of reproduction in birds and mammals involves thyrotropin beta-chain (TSHb), which is secreted from the pars tuberalis (PT) and controls the expression of deiodinase type 2 and 3 in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHRs). To analyze the impact of melatonin and the molecular clockwork on the expression of Tshb and Tshr, we investigated melatonin-proficient C3H wild-type (WT), melatonin receptor 1-deficient (MT1-/-) or clockprotein PERIOD1-deficient (mPER1-/-) mice. Expression of Tshb and TSHb immunoreactivity in PT were low during day and high during the night in WT, high during the day and low during the night in mPER1-deficient, and equally high during the day and night in MT1-deficient mice.

The mammalian molecular clockwork controls rhythmic expression of its own input pathway components.

The core molecular clockwork in the suprachiasmatic nucleus (SCN) is based on autoregulatory feedback loops of transcriptional activators (CLOCK/NPAS2 and BMAL1) and inhibitors (mPER1-2 and mCRY1-2). To synchronize the phase of the molecular clockwork to the environmental day and night condition, light at dusk and dawn increases mPer expression. However, the signal transduction pathways differ remarkably between the day/night and the night/day transition.

Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny.

Circadian rhythms of many body functions in mammals are controlled by a master pacemaker, residing in the hypothalamic suprachiasmatic nucleus (SCN), which synchronises peripheral oscillators. The SCN and peripheral oscillators share several components of the molecular clockwork and comprise transcriptional activators (BMAL1 and CLOCK/NPAS2) and inhibitors (mPER1/2 and mCRY1/2). Here we compared the ontogenetic maturation of the clockwork in the SCN and pars tuberalis (PT).

Clock gene expression in the retina of melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice.

In several mammalian species, the retina contains an autonomous circadian clock and is capable of synthesizing melatonin. The function of circadian clocks depends on interlocking transcriptional/translational feedback loops involving several clock genes. Here we investigated the expression of two clock genes (Per1, Cry2) and the level of phosphorylated (p) cyclic AMP response element binding protein (CREB) in retinae of melatonin-deficient (C57BL) with an intact retina and melatonin-proficient (C3H) mice with degenerated outer nuclear layer.